The reactivity of sulfhydryl groups of bovine cardiac troponin C

Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84.     

Calcium-dependent protein-protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I.

The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+-dependent myofibrillar ATPase. The binding of Ca2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction.     

Internal genomic sequence of human CYP1A1 gene is involved in superinduction of dioxin-induced CYP1A1 transcription by cycloheximide

Guanylate cyclase-activating protein 2 (GCAP2) is expressed in vertebrate photoreceptors cells where it regulates the activity of membrane bound guanylate cyclases in a Ca(2+)-dependent manner. The essential trigger step involves a Ca(2+)-induced conformational change in GCAP2. We investigated these Ca(2+)-dependent changes by probing the cysteine accessibility in wild type and mutant GCAP2 forms with the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Cysteine residues in position 35 and 111 displayed a restricted accessibility in the presence of Ca(2+), whereas cysteine in position 131 reacted with DTNB in the presence and absence of Ca(2+). Our data indicate that the Ca(2+)-sensitivity of GCAP2 is significantly controlled by its third Ca(2+)-binding site, EF-hand 3.     

Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src

pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07 x 10(-9)M (KD1) and 3.93 x 10(-6)M (KD2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3A smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.     

Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue

It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.     

The primary structure of a new Mr 18,000 calcium vector protein from amphioxus

The amino acid sequence of a new Ca2+-binding protein (CaVP) from Amphioxus muscle (Cox, J. A., J. Biol. Chem. 261, 13173-13178) has been determined. The protein contains 161 amino acid residues and has a molecular weight of 18,267. The N terminus is blocked by an acetyl group. The two functional Ca2+-binding sites have been localized based on homology with known Ca2+-binding domains, on internal homology and on secondary structure prediction, and appear to be the domains III and IV. The C-terminal half of CaVP, which contains the two Ca2+-binding sites, shows a remarkable similarity with human brain calmodulin (45%) and with rabbit skeletal troponin C (40%). Functional domain III contains 2 epsilon-N-trimethyllysine residues in the alpha-helices flanking the Ca2+-binding loop. Sequence determination revealed two abortive Ca2+-binding domains in the N-terminal half of CaVP with a similarity of 24 and 30% as compared with calmodulin and troponin C, respectively. This half is also characterized by the presence of a disulfide bridge linking the N-terminal helix of domain I to the C-terminal helix of domain II. This disulfide bond is very resistant to reduction in the native state, but not in denatured CaVP. The optically interesting aromatic chromophores (2 tryptophan and 1 tyrosine residues) are all located in the nonfunctional domain II.     

Salt-induced conformational changes in skeletal myosin light chains, troponin-C, and parvalbumin

Evidence for salt-induced changes in myosin light chains [dissociated by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)], troponin-C (TnC), and parvalbumin was obtained from chymotryptic digestion, circular dichroism, fluorescence, and difference absorption studies. High salt (0.6 M NaCl) protects the DTNB light chain from proteolysis, increases its alpha-helical content, and quenches the tryptophan fluorescence. These effects are similar to the changes induced by Ca2+ but smaller in magnitude. TnC is affected by monovalent cations in a similar manner. Changes in the alpha-helical content resemble the effect of Ca2+. The enhancement of tyrosine fluorescence reflects conformational changes in the Ca2+-Mg2+ binding sites. The increase in the fluorescence of dansylaziridine-labeled TnC suggests perturbation of Ca2+-specific sites by salt. Cancellation of this effect by Mg2+ binding to the high-affinity sites is indicative of site-site interactions. In Whiting parvalbumin, salt-induced a perturbation of tryptophan absorption similar in nature to the Ca2+ effect.     

cDNA cloning of human calpastatin: sequence homology among human, pig, and rabbit calpastatins

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.     

Cys32 and His105 are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter

CvaB, a member of the ATP-binding cassette transporter superfamily, is the central membrane transporter of the colicin V secretion system in Escherichia coli. Cys32 and His105 in the N-terminal domain of CvaB were identified as critical residues for both colicin V secretion and cysteine proteolytic activity. By inhibiting degradation with N-ethylmaleimide and a mixture of protease inhibitors, a stable wild-type N-terminal domain (which showed cysteine protease activity when activated) was purified. Such protease activity was Ca2+- and concentration-dependent and could be inhibited by antipain, N-ethylmaleimide, EDTA, and EGTA. At low concentrations, the Ca2+ analogs Tb3+ and La3+ (but not Fe3+) significantly enhanced proteolytic activity, suggesting that the size of the cations is important for activity. Together with comparisons of the sequences of members of the cysteine protease family, these results indicate that Cys32 and His105 are the critical residues in the CvaB N-terminal domain for the calcium-dependent cysteine protease activity and secretion of colicin V.     

Contribution of distinct structural elements to activation of calpain by Ca2+ ions

The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a K(d) of 3400 microm. Phospholipids lower this value to 220 microm. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mm for Calpain B and from 250 to 7 microm for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this "extended transducer" mechanism is presented.     

Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.     

The purification and characterisation of m-calpain from ostrich brain

Calpains are intracellular cysteine proteases activated in a Ca(2+)-dependent manner. The purpose of the present study was to investigate the physico-chemical and kinetic properties of ostrich brain m-calpain. m-Calpain was purified by successive chromatographic steps on Toyopearl-Super Q 650s and Pharmacia Mono Q HR 5/5 columns. A Ca(2+) concentration of 5mM and a casein concentration of 5mg/ml were found to be necessary for optimum calpain activity. Ostrich m-calpain exhibited a M(r) of 84K using SDS-PAGE and a M(min) of 79.3K from amino acid analysis. The pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. The amino acid composition of m-calpain revealed 700 residues. The N-terminal sequence of m-calpain showed sequence identity with chicken (27%), human (23%) and rabbit (18%) and Schistoma mansoni (9%).     

Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation

We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatory domain of troponin C (TnC) so we could investigate their role in troponin-linked Ca2+ regulation of muscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnC which, when Ca2+ binds to regulatory sites in this domain, is exposed and interacts with the inhibitory region of troponin I (TnI). We constructed three double mutants (E53A/E54A, E60A/E61A, and E85A/D86A) and two single mutants (R44A and R81A) of rabbit fast skeletal muscle troponin C (TnC) in which the charged residues were replaced with neutral alanines. All five of these mutants retained TnC's ability to bind TnI in a Ca2+-dependent manner, to neutralize TnI's inhibition of actomyosin S1 ATPase activity, and to form a ternary complex with TnI and troponin T (TnT). Ternary complexes formed with TnC(R44A) or TnC(R81A) regulated actomyosin S1 ATPase activity normally, with TnI-based inhibition in the absence of Ca2+ and TnT-based activation in the presence of Ca2+. TnC(E53A/E54A) and TnC(E85A/D86A) interacted weakly with TnT, as judged by native gel electrophoresis. Ternary complexes formed with these mutants inhibited actomyosin S1 ATPase activity in both the presence and absence of Ca2+, and did not undergo Ca2+-dependent structural changes in TnI which can be detected by limited chymotryptic digestion. TnC(E60A/E61A) interacted normally with TnT. Its ternary complex showed Ca2+-dependent structural changes in TnI, inhibited actomyosin S1 ATPase in the absence of Ca2+, but did not activate ATPase in the presence of Ca2+. This is the first demonstration that selective mutation of TnC can abolish the activating effect of troponin while its inhibitory function is retained. Our results suggest the existence of an elaborate network of protein-protein interactions formed by TnI, TnT, and the N-terminal domain of TnC, all of which are important in the Ca2+-dependent regulation of muscle contraction.     

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II

One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.     

Isolation and sequence of a cDNA clone for rabbit fast skeletal muscle troponin C. Homology with calmodulin and parvalbumin

The binding of Ca2+ to troponin C (TnC) regulates skeletal muscle contraction. We have isolated a full-length cDNA clone for fast skeletal muscle TnC from a neonatal rabbit skeletal muscle library and determined its nucleic acid sequence. The amino acid sequence deduced from this clone matches the previously reported amino acid sequence (Collins, J. H., Greaser, M. L., Potter, J. D., and Horn, M. J. (1977) J. Biol. Chem. 252, 6356-6362) except at the amino terminus. According to the nucleotide sequence, the first 2 residues of TnC are threonine-aspartic acid, which is the reverse of the order reported previously. The isolation of the adult form of TnC from a neonatal library suggests that there may be no developmental isoforms of fast TnC. The protein coding region of the fast TnC clone has 67% homology with the reported nucleotide sequence for chicken slow TnC (Putkey, J. A., Carroll, S. L., and Means, A. R. (1987) Mol. Cell. Biol. 7, 549-1553). The homologies between the nucleotide sequences of TnC, calmodulin, and parvalbumin provide evidence that all three proteins were derived from a common precursor molecule which had four Ca2+-binding sites.     

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.     

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.     

Studies on the structure of lecithin:cholesterol acyltransferase (LACT)--comparisons of the active site region and secondary structure of the human and the porcine enzymes

1. Chemical modification of essential serine, histidine and cysteine residues of porcine LCAT were accompanied by loss of enzymatic activity. 2. Modification of cysteine with DTNB inactivated the enzyme which could not be reactivated by KCN suggesting direct involvement of the cysteine residue(s) in catalysis. 3. About half of the primary structure of the porcine enzyme was determined. 4. Respective regions of the human and porcine LCAT are highly homologous; especially, the amino-terminus and the region surrounding the DFP-labeled serine residues. 5. The observed primary structure differences represent amino acid substitutions that are projected to induce significant changes in secondary structure.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

A calcium-43 NMR study of calcium binding to an acidic proline-rich phosphoprotein from human saliva

The 43Ca NMR line width measured for Ca2+ bound to protein A, an acidic proline-rich salivary protein, is 1 order of magnitude narrower than has previously been observed for other proteins of similar molecular weight. The correlation times, quadrupole coupling constants, and chemical shifts estimated for Ca2+ ions bound to the intact protein (Mr approximately 10 000) and its 30 amino acid residue long acidic N-terminal TX peptide were indistinguishable within experimental error. These results--as well as the outcome of 1H NMR relaxation rate measurements--are indicative of extensive motions for the protein residues, which in turn give rise to a high degree of flexibility for the protein-bound Ca2+. Ca2+ titration and pH-dependent measurements on protein A, the TX peptide, and the dephosphorylated TX peptide established the importance of the two phosphoserine residues in the binding of Ca2+. Moreover, a comparison of the 43Ca NMR parameters with those obtained for other Ca2+-binding proteins suggests the presence of Ca2+-binding sites of similar symmetry in all these proteins. No evidence was found for a proposed interaction between the highly acidic N-terminal and the weakly basic C-terminal regions of protein A. In contrast, the high pH inflection that was observed in the pH titration curve for the intact protein was also found for the phospho and dephospho TX peptides, thus suggesting that basic moieties in the N-terminal region rather than those in the C-terminal region may be responsible for this observation.